Antibodies specifically bind to substances called antigens, and detoxify and remove antigen-containing factors with the cooperation of other biomolecules and cells. The name “antibody” is particularly based on such a binding ability to an antigen, and these substances are known as “immunoglobulins”.
Recent developments in genetic engineering, protein engineering, and cell technology have led to the accelerated development of antibody drugs, which refer to pharmaceuticals utilizing the abilities of antibodies. Since the antibody drugs more specifically attack a target molecule than conventional pharmaceuticals, use thereof is expected to further reduce side effects and to produce higher therapeutic effects. In fact, these drugs contribute to improvement in various disease conditions.
The quality of antibody drugs is thought to largely depend on the purity compared to the quality of other recombinant protein pharmaceuticals because the doses of these antibody drugs to the body are very large. In order to produce a high purity antibody, techniques using an adsorbing material that contains a molecule capable of specifically binding to an antibody as a ligand (e.g. affinity chromatography) are commonly employed.
Most of the antibody drugs developed so far are monoclonal IgG antibodies. These antibodies are mass produced by recombinant cell-culture technology or the like and purified using proteins having affinities for IgG antibodies. One well-known example of immunoglobulin-binding proteins having affinities for IgG antibodies is Protein A. Protein A is a cell wall protein produced by the gram-positive bacteria Staphylococcus aureus and contains a signal sequence S, five immunoglobulin-binding domains (E domain, D domain, A domain, B domain and C domain) and an XM region, which is a cell wall-anchoring domain (Non-Patent Document 1). In an initial purification process (capture process) in antibody drug manufacture, affinity chromatography columns that contain as a ligand Protein A immobilized on a water-insoluble carrier (hereinafter, referred to as protein A columns) are commonly used (Non-Patent Documents 1 to 3).
Various techniques for improving the performance of protein A columns have been developed. Various technological developments in ligands have also been made. Initially, wild-type protein A was used as a ligand, but currently a recombinant Protein A altered by protein engineering is used as a ligand in many techniques for improving the performance of columns.
Typical examples of such a recombinant Protein A include a recombinant Protein A without the XM region that does not bind to immunoglobulins (rProtein A Sepharose (trademark) available from GE health care, Japan). Currently, columns containing as a ligand a recombinant Protein A without the XM region are widely used for industrial purposes because these columns advantageously further reduce non-specific adsorption of proteins compared to conventional ones.
Further, the use of a recombinant Protein A containing a mutant Cys residue (Patent Document 1) or a recombinant Protein A containing a plurality of mutant Lys residues (Patent Document 2) as a ligand has also been proposed. These Protein A mutants are efficient in their immobilization on a water-insoluble carrier and have advantages in the antibody-binding capacity of columns and reduction in leakage of the immobilized ligands.
Also well known is a technique using, as a ligand of an engineered recombinant Protein A, an engineered domain obtained by introducing mutation into the B domain (this engineered domain is referred to as a Z domain) (Patent Document 3, and Non-patent Documents 1 and 4). Specifically, the Z domain is an engineered domain obtained by introducing a mutation to replace the Gly residue at position 29 of the B domain with Ala. Although, in the Z domain, the Ala residue at position 1 of the B domain is also replaced with Val, this mutation is intended to facilitate genetic engineering preparation of a gene encoding multiple connected domains and does not affect the domain functions (for example, a mutant in which the Val residue at position 1 of the Z domain is replaced with Ala is used in an example of Patent Document 4)
The Z domain is known to be more alkali resistant than the B domain and advantageously can be reused through alkali washing. Patent Documents 5 and 6 disclose a ligand derived from the Z domain in which an Asn residue is replaced with another amino acid so as to impart further higher alkali resistance, and this ligand is already used for the industrial purpose.
Another feature of the Z domain is its reduced binding ability to the Fab region of immunoglobulins (Non-Patent Document 5). This feature advantageously facilitates dissociation of an antibody binding to the Z domain using an acid (Non-Patent Document 1 and Patent Document 7).
In addition to the Z domain derived from the B domain, highly alkali-resistant engineered Protein A ligands derived from the C domain of Protein A have also been studied (Patent Document 4). These ligands characteristically take advantage of the inherent high alkali resistance of the wild-type C domain and have been receiving attentions as new alternative base domains to the Z domain. However, our studies on the C domain have revealed the disadvantage that it is difficult to dissociate an antibody binding to the C domain using an acid. The C domain, as taught in Non-Patent Document 2 and Patent Document 4, has strong binding ability to the Fab region of immunoglobulins, and this ability is presumably supposed to make it difficult to dissociate the antibody using an acid. In order to overcome this disadvantage, we examined a C-domain mutant in which the Gly residue at position 29 is replaced with Ala, for its antibody dissociation properties in the presence of an acid. The result revealed that the ability of the C domain mutant was improved over that of the wild-type C domain but was still not enough. The reason for this was revealed by an analysis of the interaction between the protein molecules, and specifically was that the C domain in which the Gly residue at position 29 was replaced with Ala did not have sufficiently reduced binding ability to the Fab region of immunoglobulins.
As described above, it is widely known that replacement of the Gly residues corresponding to position 29 in the immunoglobulin-binding domains (E, D, A, B and C domains) of Protein A with Ala is a useful mutation strategy. In fact, the technologies for engineered Protein A developed after the disclosure of the “G29A” mutations in 1987 include these mutations (Patent Documents 2, 4 and 6).
However, all these technologies teach only the G29A mutations, that is, replacement of the Gly residues corresponding to position 29 with Ala as mutations of the amino acid residues corresponding to position 29 in the immunoglobulin-binding domains of Protein A and are silent about a mutation to introduce an amino acid residue other than Ala into this position. The G29A mutations are designed to minimize the conformational change. In this strategy, Ala is regarded as the best amino acid because Ala has the second smallest side chain next to Gly. Replacement with an amino acid having a larger side chain has not been examined so far (Non-Patent Document 4). Accordingly, it was unclear whether replacement of the Gly residues corresponding to position 29 with an amino acid having a larger side chain than Ala, which would result in a larger conformational change and might impair the original abilities (e.g. binding ability to immunoglobulins), could produce a better effect than that achieved by the replacement with Ala. Although it is in 1987 (Non-Patent Document 4) when replacement of the Gly residues corresponding to position 29 with Ala was disclosed, replacement with an amino acid other than Ala has not been proposed so far.
Patent Document 1: U.S. Pat. No. 6,399,750
Patent Document 2: JP 2007-252368 A
Patent Document 3: U.S. Pat. No. 5,143,844
Patent Document 4: JP 2006-304633 A
Patent Document 5: EP 1123389
Patent Document 6: WO 03/080655
Patent Document 7: U.S. Patent Application No. 2006/0194950
Non-Patent Document 1: Hober S. et al., “J. Chromatogr. B” 2007, vol. 848, pages 40-47
Non-Patent Document 2: Low D. et al., “J. Chromatogr. B”, 2007, vol. 848, pages 48-63
Non-Patent Document 3: Roque A. C. A. et al., “J. Chromatogr. A”, 2007, vol. 1160, pages 44-55
Non-Patent Document 4: Nilsson B. et al., “Protein Engineering”, 1987, vol. 1, pages 107-113
Non-Patent Document 5: Jansson B. et al., “FEMS Immunology and Medical Microbiology”, 1998, vol. 20, pages 69-78